Peanut allergy is considered to be a major health issue with global effects. To date, no effective curative approach has been applied for the therapy of the anaphylaxis resulting from the peanut allergens. The accurate and effective detection methods for the surveillance of allergens in food are still the primary strategies to avoid allergic diseases. In this study, nanobodies (Nbs) derived from the Heavy-Chain only Antibodies (HCAbs) were selected against the general peanut protein extract through the unbiased strategy to facilitate the development of the sandwich ELISA for the detection and surveillance of peanut allergen contamination. The target antigen of the selected Nb was identified as peanut allergen Ara h 3, and a cross-reaction was observed with the member of Gly 1 from the Ara h 3 family. The applicability of the self-paired Nb P43 on the establishment of the immuno-assay was verified. A sandwich ELISA against peanut allergen was developed, which reached a linear range of 0.2-10.6 μg/mL, and a limit of detection of 53.13 ng/mL.